Nuclear functions in cells depend upon critical thiols that are subject to oxidative damage by environmental, occupational and therapeutic chemicals. Thioredoxin-1 (Trx1) and glutathione (GSH) are biological thiols that protect against oxidants. These thiols also function in redox signaling and control. The purpose of this proposal is to gain an understanding of the distinct functions of Trx1 and GSH in protection against oxidative stress in cell nuclei. Aim 1 is focused on the quantitative importance of the nuclear Trx1 system compared to cytoplasmic Trx1 and GSH-dependent systems for protection against oxidative stress. Cell models will be used to examine nuclear responses to exogenous H2O2, endogenously nuclear-generated H2O2 and oxidant chemicals. Novel redox western blot and mass spectrometry proteomic techniques will be used to determine the redox state of nuclear Trx1 and S-glutathionylation of nuclear proteins. Nuclear-targeted Trx1 (NLS-Trx1) and dominant negative forms will be used to control the nuclear Trx1 system;NLS-peroxiredoxin-1 (NLS-Prx1) will be used to enhance nuclear peroxide metabolism;and cellular GSH will be modified by control of synthesis and by selective depletion. Nuclear-targeted (NLS-) D- amino acid oxidase with added D-amino acids will be used to stimulate intranuclear H2O2 generation. Toxicants will be studied to test for selective nuclear resistance/sensitivity to oxidative stress. Specific Aim 2 is to develop transgenic mouse models expressing fusion proteins with nuclear localization and nuclear export signals (NES) fused to human Trx1 (NLS-hTrx1 and NES-hTrx1) and use these for in vivo toxicologic studies of nuclear oxidative stress. Transgenic mice will be characterized for nuclear and cytoplasmic redox under control conditions and with an established in vivo toxicologic challenge with the cardiotoxicant, adriamycin. The results of these studies will provide the first test of the hypothesis that enhanced nuclear antioxidant systems protect against toxicity in vivo. Specific Aim 3 is to use mass spectrometry-based redox proteomic techniques to identify nuclear proteins controlled by the Trx1 and GSH systems and to identify nuclear proteins that are selectively vulnerable to oxidative stress. The successful completion of these aims will provide fundamental new knowledge concerning the quantitative importance of major thiol/disulfide control systems in nuclei in protection against oxidative stress from environmental and therapeutic toxicities.